Dendritic cells and class II MHC expression on thyrocytes during the autoimmune thyroid disease of the BB rat.

The BB rat is a well-established model for spontaneous thyroid autoimmune disease. Since antigen presentation in thyroid autoimmunity is still a matter of debate, we studied the presence of antigen-presenting dendritic cells in the thyroid of the BB/O rat during the development of the disease in relation to the presence of other leucocytes and the aberrant expression of class II MHC determinants by thyrocytes. Thyroid glands, as well as thyroid-draining lymph nodes, were investigated in enzyme histochemistry and immune histochemistry. The appearance of anti-colloid antibodies in the circulation at 6 weeks of age was accompanied by an increase in the weight of the thyroid-draining cervical lymph nodes, which contained many anti-thyroglobulin-producing plasma cells. The only noteworthy event in the thyroid gland in this early stage of the disease was an increase in the number of dendritic cells. T cells, B cells, and plasma cells were virtually absent from the thyroid, and thyrocytes were invariably negative for class II MHC determinants. Only after 18 weeks of age, when large accumulations of dendritic cells, B lymphocytes, and T lymphocytes were seen in 40% of the BB thyroids, could some class II MHC positive thyroicytes be observed. At this stage the thyroid also contained some anti-thyoglobulin-producing plasma cells. Our observations suggest that dendritic cells play a role in antigen presentation in the early stages of the thyroid autoimmune response.

Antibodies to 'second colloid antigen.' A study on the prevalence in sporadic forms of congenital hypothyroidism.

Antibodies against the so called 'second colloid antigen' (CA2 antibodies) occurred in 51% of the mothers of hypothyroid children detected by screening for neonatal congenital hypothyroidism in Quebec (N = 49) and in The Netherlands (N = 26). In The Netherlands where corresponding neonatal serum was available, 31% (8 of 26) of the infants with congenital hypothyroidism were positive for antibodies against the second colloid antigen. When during follow-up, 3 to 5 years after diagnosis, the mothers and their children were investigated, 46% (7 of 15) of the mothers were positive for antibodies against the second colloid antigen, whereas 29% (4 of 14) of the hypothyroid children were also positive. Various control groups did not show more than a 12% positivity. This presence of thyroid-reactive antibodies in a proportion of the hypothyroid children 3 to 5 years after diagnosis is not compatible with a mere transplacental passage; it indicates that the antibodies must be produced by the mothers and by the children themselves. We conclude that a thyroid autoimmune response occurs in a considerable part of infants with congenital hypothyroidism and their mothers and that this immune response seems to persist in both of them for years.

Thyroid autoimmunity in endemic goitre caused by excessive iodine intake.

The pathophysiology of endemic goitre caused by excessive iodine intake is not well defined. By interacting with the immune system, iodine excess may trigger the development of autoimmune thyroid disease such as lymphocytic Hashimoto's thyroiditis (LT). In an attempt to examine this further, we compared the presence of thyroid autoantibodies in 29 goitrous children, from an iodine excess area, and in 26 healthy children, from an iodine sufficient area, of north central China. Serum was tested for antimicrosomal (MAb), anti-thyroglobulin (TgAb), second colloid antigen antibodies (CA2-Ab) and TSH binding inhibitory immunoglobulins (TBII). Affinity chromatographically purified IgG was tested for thyroid growth-stimulating activity (TGI) by two different methods: a sensitive cytochemical bioassay (CBA) using guinea-pig thyroid explants and a mitotic arrest assay (MAA) employing a continuous rat thyroid cell line (FRTL-5). We found no increased prevalence of LT in patients with endemic iodine goitre. The levels of MAb, TgAb and CA2-Ab did not differ significantly between the two groups of children. Further, TBII were not present in either group. Thyroid growth-stimulating immunoglobulins (TGI) were the major autoantibodies found in children with goitres caused by iodine excess. In the CBA, 12 of 20 (60%) goitrous children and 0 of 12 (0% P less than 0.05) healthy children were positive for TGI. Similar results were found in the MAA, and a good correlation between results of the CBA and MAA was found (P = 0.003). Maximal TGI activity in dose-response CBA showed a good relation with clinical goitre size (r = 0.63; P less than 0.05) indicating a possible pathophysiological role for these antibodies. We conclude that endemic iodine goitre is not associated with Hashimoto's lymphocytic thyroiditis. Nevertheless, autoimmune growth factors such as TGI may play a primary role in the pathogenesis of thyroid growth in this condition.

Endemic cretinism: possible role for thyroid autoimmunity.

Thyroid atrophy, rather than goitre, is a characteristic feature of myxoedematous cretinism but its cause and nature are unknown. In this study, purified IgG fractions of serum from patients with myxoedematous endemic cretinism inhibited thyrotropin-induced DNA synthesis in guineapig thyroid segments in a sensitive cytochemical bioassay. IgG from patients with euthyroid neurological endemic cretinism or from normal subjects did not inhibit thyroid growth. Furthermore, in myxoedematous subjects, the presence of the thyroid-growth-blocking immunoglobulins showed a positive relation with thyroid atrophy found on ultrasound. These findings provide a pathogenic basis for the variable clinical expression of endemic cretinism.

The long-term outcome of thyroidectomy for sporadic non-toxic goitre.

To study the long-term outcome after thyroidectomy, 113 sporadic non-toxic goitre patients who underwent thyroidectomy in our hospital in the period 1974-1983, were studied. Five patients complained of recurrent goitre; a goitre was found on inspection and palpation in these five and in 15 others. There were no differences between the 20 patients with goitre and the 93 patients without goitre with regard to sex, age, duration of goitre, indication and type of thyroidectomy, postoperative thyroid hormone medication, period of follow-up, and T4, T3, or TSH plasma values at the time of follow-up examination. Twenty-three patients complained of voice changes since thyroidectomy. In a case control study, included in this follow-up study, 19 patients with goitre, i.e. thyroid size I and II as estimated by inspection and palpation (cases), and 16 patients without goitre, i.e. thyroid size OA and OB (controls), were studied in more detail. No difference between cases and controls was found in any of the above mentioned parameters that could explain the recurrence of goitre. Thyroid volume (median) was greater in the cases (34.1 ml, range 7.9-83.4) than in the controls (10.3 ml, range 2.5-48.7) (P less than 0.001), although a considerable overlap between the two groups was observed. One or more thyroid nodules were found in 89.5% of the cases and in 62.5% of the controls (NS). Serum thyroid growth stimulating immunoglobulin (TGI) was present both in cases (68%) and controls (50%). TGI was present in high titres in all five patients who complained about recurrent goitre.(ABSTRACT TRUNCATED AT 250 WORDS)

The goitre of the BB/O rat: an animal-model for studying the role of immunoglobulins stimulating growth of thyroid cells.

The BB rat is a well-known animal model for the study of autoimmune thyroid disease. Antithyroglobulin antibodies can be detected in the circulation from the age of 6 weeks onwards, and accumulations of lymphoid cells occurs in the thyroid of up to 60% of animals at the age of 20 weeks and over. The rat, however, stays euthyroid, the thyroid is not destroyed, and hence the disease is not yet well characterized. The study reported here shows that the thyroid weights of 41% (40/97) BB rats were raised from week 6 onwards in comparison to those of Wistar controls. Morphologically, BB thyroids showed a strong similarity to the human disease entity 'colloid goitre', namely an active growth, high columnar epithelium, branching and budding of thyrocytes, no signs of thyroid destruction and in 42% of rats at the age of 22-26 weeks, a development of intrathyroidal lymphoid tissue. Plasma TSH was not significantly raised in the animals. For this reason the presence of immunoglobulins which stimulate the growth of thyroid cells (so-called TGI's) was determined in 12-16-week-old BB rats (n = 10) and in control Wistar rats (n = 10). At this time a significant difference could be recorded in thyroid weights between BB/O rats and Wistar controls (20.1 +/- 6.0 mg vs. 15.8 +/- 2.9 mg, respectively), even in the absence of any intrathyroidal lymphoid cell infiltration. Protein-A-Sepharose purified serum IgG of these animals was used to detect TGI-activity via the 'Feulgen Cytochemical Bioassay'. Of the BB/O rats, eight were clearly positive for TGI, all of the Wistar rats were negative. The data show that the autoimmune prone BB rat may thus serve as an animal model for euthyroid goitre associated with thyroid stimulating antibodies.

Immunoglobulins of patients with Cushing's syndrome due to pigmented adrenocortical micronodular dysplasia stimulate in vitro steroidogenesis.

The putative stimulation of adrenal steroid production by immunoglobulins (Igs) of five patients with pigmented adrenocortical micronodular dysplasia and clinical Cushing's syndrome was investigated. Ascorbate depletion, a process linked to steroid production, was measured by a cyto-chemical bioassay employing guinea pig adrenal explants in organ culture and exposed to IgG from the patients and normal subjects. We also measured cortisol production by these segments during a 5-h culture period using a RIA. For positive reference values we studied the effects of ACTH-(1-39), ACTH-(1-24), ACTH-(11-24), and ACTH-(18-39) on in vitro ascorbate depletion and cortisol production. Both ACTH-(1-39) and ACTH-(1-24) depleted ascorbate and stimulated cortisol production in adrenal cells. The dose-response kinetics of the peptides were bell-shaped; maximal responses were reached in both instances at 1 fmol/L to 10 pmol/L. In all tests, stimulation of in vitro cortisol production was paralleled by ascorbate depletion. ACTH-(18-39) also stimulated ascorbate depletion and cortisol production, but at one concentration only (100 fmol/L), and TSH and LH had no effect. Protein-A-Sepharose-purified IgG preparations of the five patients stimulated ascorbate depletion and/or cortisol production in a dose-dependent fashion; however, the responses occurred over a narrow concentration range (15-150 micrograms IgG/mL culture fluid). These observations support the hypothesis that the hypercortisolism of the syndrome of pigmented adrenocortical micronodular dysplasia is due to circulating Igs that stimulate adrenal steroidogenesis.

Intrathyroidal dendritic cells.

The presence, marker pattern, and ultrastructure of antigen-presenting dendritic cells were studied in normal thyroid glands from 9 subjects (6 obtained at surgery; 3 at autopsy) and in the thyroid glands form 13 patients with Graves' hyperthyroidism, 10 patients with simple nontoxic goiter, and 1 patient with Hashimoto's disease (all obtained at surgery). The immunohistochemical characterization of the cells was carried out using the monoclonal antibodies OKIa (class II MHC determinants), RFD1 and L25. These latter monoclonal antibodies react strongly with active dendritic cells in T-cell areas of secondary lymphoid organs (the interdigitating cells in lymph nodes and spleen). Antigen-presenting dendritic cells were defined as cells with an eccentric reniform nucleus, long cytoplasmic protrusions, and strong membrane-bound class II MHC positivity combined with little or no cytoplasmic acid phosphatase activity. According to these criteria normal human thyroid tissue contained a few dendritic cells; they were localized outside the thyroid follicles. These dendritic cells in normal thyroid tissue lacked the marker molecules identified by the monoclonal antibodies RFD1 and L25. In fact, the majority of the dendritic cells were strongly positive for the C3bi receptor (identified by the monoclonal antibody FK 24), which indicates a more monocyte/macrophage character of the cell. In Hashimoto's goiter, Graves' disease, and sporadic nontoxic goiter (which we consider an autoimmune thyroid disease) the numbers of dendritic cells were higher compared to those in the normal gland, and these dendritic cells were clearly positive for RFD1 and L25. The cells were often seen in contact with a few intrathyroidal lymphocytes, forming small lymphoid cell clusters. They were also found in the T-cell zones of larger well organized intrathyroidal lymphoid structures (focal thyroiditis). On ultrastructural examination the dendritic cells in Graves' glands, Hashimoto's goiter, and sporadic nontoxic goiter were similar to the interdigitating cells present in secondary lymphoid organs. The data suggest active involvement of dendritic cells in the immune process in the thyroids of patients with autoimmune thyroid disease.

Effects of ACTH and ACTH fragments on DNA synthesis in guinea-pig adrenal segments kept in organ culture.

Stimulation of adrenal DNA synthesis by ACTH and its fragments ACTH (Synacthen) and ACTH was investigated. Synthesis of DNA was measured as the increase in the percentage of cells in S-phase (Feulgen densitometry) in guinea-pig adrenal explants kept in organ culture and exposed to the peptides for 5 h at 37 degrees C. ACTH and its C-terminal fragment ACTH (corticotrophin-like intermediate lobe peptide) were found to be potent stimulators of in-vitro adrenal DNA synthesis. The dose-response kinetics were biphasic and optimal responsiveness was reached in both instances at 1 fmol/1-10 pmol/1 (this biological effect of ACTH has hitherto not been described). The N-terminal fragment ACTH gave only minimal responses. Thyrotrophin and LH, tested as controls, did not induce adrenal DNA synthesis. Epidermal growth factor was a potent stimulator of adrenal DNA synthesis in vitro. Our data suggest a trophic action of the C-terminal part of the corticotrophic molecule. Clear trophic effects were also found for the N-terminal part of the pro-opiomelanocortin molecule N-POC (optimum 0.1 nmol/l) and N-POC(51-62) (optimum 0.1 pmol/l). The latter observations support earlier concepts that this part of the pro-opiomelanocortin molecule has a stimulatory effect on adrenal DNA synthesis.

Dendritic cells in autoimmune thyroid disease.

Dendritic cells form a morphologically distinct class of cells characterized by shape, reniform nucleus, absent to weak acid-phosphatase activity and strong Class II MHC determinant positivity. Functionally they are the most efficient cells in antigen presentation to T-lymphocytes which indicates their role in the initiation of an immune response. Using immunehistochemical techniques we studied the presence of dendritic cells in normal Wistar rat and human thyroids, in thyroids of BBW rats developing thyroid autoimmunity and in Graves' goitres. Dendritic cells could be identified in all thyroids studied and were positioned underneath the thyrocytes in between the follicles. Skin dendritic cells travel via lymphatics to draining lymph nodes, thus forming an antigen presenting cell system. It is likely that a similar cell system exists on the level of the thyroid for dendritic cells have also been detected in thyroid draining lymph nodes. In normal thyroid tissue of both human and rat dendritic cells were relatively scarce. During the initial phases of the thyroid autoimmune response in the BBW rat (before the appearance of Tg-antibodies in the circulation) numbers of thyroid dendritic cells increased. Intrathyroidal T-helper cells, B-cells or plasma cells could not be found. The thyroid draining lymph node contained large numbers of plasma cells. During the later stages of the thyroid autoimmune response in the BB/W rat (after the appearance of Tg-antibodies in the circulation) and in Graves' goitres dendritic cells were not only present in high number, but 20-30% were seen in contact with now-present intrathyroidal T-helper lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

T-suppressor cell defects in euthyroid nonendemic goitre.

Recently it has been suggested that a substantial number of nonendemic goitre cases can be considered as organ-specific autoimmune disorders of the thyroid. Circulating immunoglobulins, probably receptor autoantibodies, stimulating guinea pig thyroid growth in vitro (TGI) can be found in 2/3 of such patients. Defects in the regulatory balance between T-helper (Th) and T-suppressor (Ts) cells have been described in other thyroid autoimmune diseases, such as Graves' disease and Hashimoto goitre. Such defects are thought to play a role in the loss of control over thyroid autoantibody producing B cells. To investigate whether Ts cell defects can also be found in nonendemic euthyroid goitre, we studied their number and function in 15 of such patients. All patients were clinically euthyroid. Eleven were positive for TGI. Circulating T cells, Th cells and Ts/cytotoxic cells were enumerated using monoclonal antibody techniques (OKT3, Leu3a and OKT8, respectively). Suppressor cell function was assessed employing a proliferation assay in which a short-lived population of such cells was removed by a 24 h preculture. A significant difference was found between patients and controls regarding the Leu3a+/OKT8+ cell ratio: 2.74 (SD 0.94) in patients vs 1.75 (SD 0.38) in controls (P less than 0.01); the disturbed ratio was mainly due to a decrease in the percentage of OKT8+ cells. The functional Ts cell assay also showed a defect of patient lymphocytes: patient removal index (SRI) was 1.5 (SD 1.0) vs an index of 2.6 (SD 1.2) for healthy controls (P less than 0.05). A fair correlation between the numerical and functional data on Ts cells was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Familial Cushing's syndrome due to nodular adrenocortical dysplasia. A putative receptor-antibody disease?

Two sisters aged 13 and 19 years suffering from familial Cushing's syndrome due to nodular adrenocortical dysplasia are described. Pituitary adrenocortical function tests indicated the presence of adrenal autonomy. Adrenal scintigraphy showed bilateral symmetrical uptake indicating the bilateral character of the autonomous process. Complete adrenalectomy was performed in both girls. The adrenals were of about normal weight showing numerous dark brown pigmented nodules and small perivascular lymphocytic infiltrates. Serum immunoglobulin preparations obtained from both girls stimulated adrenocortical cell growth in a cytochemical bioassay system. It is proposed that circulating growth factors may be involved in the pathogenesis of the disease.

Congenital hypothyroidism in a Turkish family: the role of immunoglobulins blocking the trophic effects of TSH and maternal-foetal relationship.

A Turkish family with frequent intermarriages is described, in which two siblings were born with persistent forms of congenital hypothyroidism, in the elder child concomitant with absent radioactive thyroid imaging. The mother was clinically euthyroid throughout the period of observation, but showed in addition to thyroid microsomal antibodies, high levels of immunoglobulins blocking the trophic action of TSH. These maternal growth blocking antibodies were transiently present in the youngest of the siblings (from birth to 2 months of age). She had a relatively mild form of congenital hypothyroidism (T3: 33 micrograms/100 ml; T4: 3.9 micrograms/100 ml). The older sibling, with proven non-functioning thyroid tissue (negative thyroidscan, T4: 0.4 microgram/100 ml) produced the growth-blocking immunoglobulins herself and may thus represent a juvenile form of thyroid autoimmunity with a very early onset. An aunt and uncle of the children, both hypothyroid since birth, were at the age of 19 and 18 years weakly positive for growth blocking immunoglobulins. This study indicates that familial forms of congenital hypothyroidism are probably complex and may be brought about by maternal to foetal passage of thyroid reactive autoantibodies, but also by the inheritance of a trait for thyroid autoimmunity. In some cases these two mechanisms might act in conjunction.

Autoimmunity and the pathogenesis of localized thyroid autonomy (Plummer's disease).

The pathogenesis of Plummer's disease (localized thyroid autonomy) is incompletely known. It has been shown that a normal thyroid follicle can harbour cells with widely different properties, and hyperactive follicles may arise from cells with both high replicating and hormonogenic potencies under the influence of chronic mild stimulation. We now find thyroid growth-stimulating immunoglobulins (TGI) to be present in low or intermediate titre in the serum of 7 of 9 patients with Plummer's disease. Haemagglutinating antibodies against thyroid microsomal antigen were present in low titre in two of these as well as in three of 21 other patients with this disorder. Two of the patients had a suppressible goitre in addition to the autonomous nodule. One of these is described in more detail. The possibility is discussed that autoimmunity may play a pathogenic role in Plummer's disease. TGI, in relatively low titres as found here, could exert the chronic mild stimulation supposed to be the prime event in the generation of hyperactive follicles. Whether autonomy is intrinsically present in these follicles or triggered by stimulation, remains to be established. Hyperthyroidism supervenes only when the mass of autonomous cells surpasses a certain limit. It appears that these patients with Plummer's disease should be included in the multidimensional spectrum of autoimmune thyroid disease.

Further studies on thyroid growth-stimulating immunoglobulins in euthyroid nonendemic goiter.

Sixty-two consecutive patients with sporadic euthyroid goiter (57 women and 5 men) from noniodine-deficient areas, including 15 patients with diffuse goiter, 39 patients with multinodular goiter, and 8 patients with a single nodule, were studied for the presence of serum thyroid growth-stimulating immunoglobulins (TGI) by the ultrasensitive cytochemical bioassay based on DNA cytophotometry (Feulgen-cytochemical bioassay). Using strictly specified conditions, 43 patients (67%) were positive. Values tended to be high in diffuse goiter, nodular goiters reccuring after partial thyroidectomy, and those with recent growth. Thirty-seven individual immunoglobulin (Ig)-rich fractions obtained by ammonium-sulfate precipitation from 20 normal subjects, 13 atrophic thyroiditis patients, and 4 dyshormonogenetic goiter patients were tested similarly, and only 3 gave positive growth assays. These results lend further support to our concept that a majority of patients with sporadic nontoxic simple goiters have a variant of thyroid autoimmune diseases separate from lymphocytic thyroiditis. With regard to assays thought to reflect activities of TSH receptor antibodies, none of 20 tested Ig preparations stimulated thyroid cAMP production. The TSH binding inhibition assay gave weak positive activity, but failed to correlate with either TGI or TSH unresponsiveness to TRH. These findings suggest that sporadic goiter TGI is not directed to the TSH-binding site. Dose-response studies performed with Igs of patients with nontoxic goiters and with human TSH standard and goitrous hyperthyroid Graves Igs as controls all revealed bell-shaped responses. Similar maximal values were reached regardless of the growth stimulus applied. However, approximately 10 times more Ig was needed to reach maximal responsiveness in sporadic goiter than in goitrous Graves' disease (i.e. 125-500 micrograms vs. 15-125 micrograms Ig/ml culture fluid). The optimal dose of human TSH ranged from 0.01-1.0 microU/ml. The assays in the present series of the 62 consecutive patients with nontoxic diffuse or nodular goiter were all carried out with a fixed amount of 125 micrograms Ig/ml and considering a value above 5% of cells in the S-phase as a positive assay. Some Ig preparations negative for TGI at this concentration may contain TGI when tested using other doses, and these are a prerequisite to assess the potency of growth antibodies in individual patients.

Role of maternal immunoglobulins blocking TSH-induced thyroid growth in sporadic forms of congenital hypothyroidism.

Among 34 mothers of infants with sporadic congenital hypothyroidism detected in the Quebec screening programme, 15 had immunoglobulins blocking thyroid growth induced by thyroid-stimulating hormone (TSH) when tested in the sensitive Feulgen cytochemical bioassay. At the time of delivery all the mothers were clinically and biochemically euthyroid, and in general the growth-blocking immunoglobulins were found in the absence of thyroid antimicrosomal antibodies. 2 mothers, however, had significant titres of antimicrosomal antibodies. They became hypothyroid 1 and 3 years after delivery. 8 of 16 post-partum infant blood samples were positive for immunoglobulins blocking TSH-induced thyroid growth. 4 of 7 positive mothers tested up to 3 years after delivery had become negative, thus indicating a trend for these immunoglobulins to disappear from the maternal circulation. Thus, transplacental passage of maternal immunoglobulins influencing TSH-induced processes of thyroid growth may play a part in the pathogenesis of sporadic congenital hypothyroidism.

In vitro trophic effects of synthetic ACTH1-24.

In vitro trophic effects of adrenocorticotrophin1-24 (ACTH1-24, Synacthen) on adrenal cells were studied, using an in vitro assay system of guinea-pig adrenal segments kept in organ culture. Two separate methods for detecting growth activity were used, namely the measurement of thymidine kinase and a nucleic acid cytophotometric method. Synthetic ACTH was able to induce growth in the adrenal explants at very low concentrations (10-25 fg ml-1). Biphasic dose-response curves were obtained, comparable to those described for other cytochemical bioassays. The principles of this assay system may allow the development of a new bioassay for the measurement of plasma concentrations of ACTH or antibodies mimicking the growth effect of this trophic hormone.

Application of in-vitro techniques to determine proliferation in human sarcoid lymph nodes.

Sarcoidosis is suggested to be a granulomatous disease with high-turnover characteristics. In this paper the turnover of the inflammatory cells (the epithelioid cells) in lymph node granulomas from sarcoidosis patients is described. Uniform slices of granulomatous lymph nodes were incubated with 3H-thymidine; autoradiographically the DNA-synthesizing cells, parameter for local proliferation, were detected and the L.I. (labelling index) was determined. We observed mostly low-turnover reactions, incidentally granulomas with high-turnover characteristics are found within the same lymph node. The uptake of 3H-TdR demonstrates that these granulomas could be in different stages of activation.

Nitroblue tetrazolium-dye reduction by rat peritoneal macrophages during the uptake of Diplococcus pneumoniae, type VI.

The relationship between the rate of particle ingestion and the rate of Nitroblue Tetrazolium (NBT)-dye reduction by macrophages was studied after incubation of peritoneal exudate macrophages with heat-killed type VI pneumococci. The adherence to a polyethylene surface of the macrophages during the uptake of the pneumococci was determined as well. In some experiments pneumococci opsonized with heat-stable opsonins were used as material to be ingested. The NBT-dye reduction and the surface adherence of the macrophages was enhanced when ingesting normal heat-killed pneumococci. During the uptake of opsonized, heat-killed pneumococci the macrophages showed an unaltered NBT-dye reduction and less adherence to a polyethylene surface as compared with macrophages incubated with normal heat-killed pneumococci. This implies that using opsonized pneumococci the quantitative NBT-dye reduction assay is not reliable as a parameter for macrophage phagocytosis, because the uptake was in fact enhanced. The surface adherence of the macrophages did not reflect the enhanced ingestion of opsonized bacteria either.